Analysis Of Drought-tolerant Transcriptome And Cloning And Characterization Of Drought Tolerance Associated Genes IbWRKY2,IbGATA24 And IbSDT In Sweetpotato(Ipomoea Batatas(L.)Lam.)

Sweetpotato,Ipomoea batatas(L.)Lam.,is an important crop for food,feed,industrial materials and bio-energy resource.Its growth and productivity are seriously limited by drought and salt stresses.In this study,the transcriptome of the drought-tolerant sweetpotato line ’Xu55-2’ under PEG6000 stress was conducted to identify the molecular mechanism involved in drought stress responses.The differentially expressed ESTs were analyzed,and IbWRKY2,IbGATA24 and IbSDT genes associated to drought and salt stresses tolerance were cloned.The functions of IbWRKY2,IbGATA24 and IbSDT were characterized by the Agrobacterium tumefaciens-mediated transformation system of Arabidopsis.The main results are as follows:1.Systematic characterization and comparison of drought tolerance were performed between‘Xu55-2’and ’Shangshu9’.’Xu55-2’ with higher drought tolerance was used for transcriptome sequencing under PEG6000 stress.Transcriptome analysis of‘Xu55-2’was conducted on Illumina HiSeq 2500 platform.A total of 86.69 Gb clean data were generated and assembled into 2,671,693 contigs,222,073 transcripts and 73,636 unigenes.In total,11,359 differentially expressed genes(DEGs)were identified after PEG6000 treatment,of which 7,666 were up-regulated and 3,693 were down-regulated.Of the 11,359 DEGs,10,192 DEGs were annotated in at least one database,and the remaining 1,167 DEGs were unknown.Abscisic acid(ABA),ethylene(ETH)and jasmonic acid(JA)signalling pathways play a major role in drought tolerance of sweetpotato.Drought-inducible transcription factors were identified,some of which have been reported to be associated with drought tolerance and others are unknown in plants.In addition,7,643 SSRs were detected.This study not only reveals insights into the molecular mechanism of drought tolerance in sweetpotato but also provides the candidate genes involved in drought tolerance of this crop.2.According to the differentially expressed EST obtained from transcriptome analysis of ’Xu55-2’under PEG6000 stress,a novel WRKY transcriptional factor gene IbWRKY2 was cloned with RACE method.Its full-length ORF contains 2,133 bp that encodes a 710-amino acid polypeptide with a molecular weight of 76.38 kDa and pI of 5.84.The genomic sequence of IbWRKY2 is 2,815 bp containing 5 exons and 4 introns.The IbWRKY2 promoter exhibited the activity and was induced by drought or salt stress in Arabidopsis.The expression of IbWRKY2 was significantly induced by salt,drought and ABA.The IbWRKY2 protein was localized to nucleus.IbWRKY2 exhibited transactivation activity and its transactivation domain was located in the N-terminal.The IbWRKY2-overexpressing Arabidopsis plants exhibited significantly higher salt and drought tolerance compared with the wild-type(WT).The significant increase of ABA,JA and proline contents and SOD activity and significant reduction of MDA and H2O2 contents were found in the transgenic Arabidopsis plants comparing with WT under drought or salt stresses.qRT-PCR analysis showed that overexpression of IbWRKY2 up-regulated the genes involved in ABA signalling pathway,proline synthesis pathway and ROS-scavenging system under salt and drought stresses.In addition,yeast two-hybrid assay(Y2H)and bimolecular fluorescence complementation assay(BiFC)indicated that IbWRKY2 could interact with VQ4 protein which regulated drought and salt stress tolerances together in plants.3.According to the differentially expressed EST obtained from transcriptome analysis of‘Xu55-2’under PEG6000 stress,a novel GATA transcriptional factor gene IbGATA24 was cloned with RACE method.The full-length ORF of IbGATA24 contains 1,059 bp that encodes a 352-amino acid polypeptide with a molecular weight of 38.58 kDa and pI of 5.62.Its genomic sequence is 2,672 bp containing 10 exons and 9 introns.β-Glucuronidase analysis in Arabidopsis showed that the IbGATA24 promoter had the activity and was induced by drought or salt stress.qRT-PCR analysis revealed that the expression of IbGATA24 was significantly induced by salt,drought and ABA treatments.The subcellular location indicated that IbGATA24 was localized to nucleus.IbGATA24 exhibited no transactivation activity in yeast.The IbGATA24-overexpressing Arabidopsis plants exhibited significantly higher salt and drought tolerance compared with WT.The significant increase of ABA,JA,BR and proline contents and SOD activity and significant reduction of MDA and H2O2 contents were found in the transgenic Arabidopsis plants comparing with WT under drought or salt stress.qRT-PCR analysis showed that overexpression of IbGATA24 up-regulated the genes involved in ABA and JA signalling pathway and ROS-scavenging system under salt and drought stresses.In addition,Y2H and BiFC demonstrated that IbGATA24 could interact with GATA24 and COP9-5a proteins which regulated drought and salt stress tolerances together in plants.4.According to the differentially expressed EST obtained from transcriptome analysis of ’Xu55-2’under PEG6000 stress,an uncharactried gene IbSDT was cloned with RACE method.The full-length ORF of IbSDT contains 621 bp that encodes a 206-amino acid polypeptide with a molecular weight of 22.04 kDa and pI of 12.10.Its genomic sequence contains only 1 exon.β-Glucuronidase analysis in Arabidopsis showed the IbSDT promoter had the activity and was induced by drought or salt stress.qRT-PCR analysis revealed that the expression of IbSDT was significantly induced by salt,drought and ABA treatments.The subcellular location indicated that IbSDT was localized to nucleus,plasma and cell membrane.IbSDT exhibited no transactivation activity in yeast.The IbSDT-overexpressing Arabidopsis plants exhibited significantly higher salt and drought tolerance compared with WT.The significant increase of ABA,JA and proline contents and SOD activity and significant reduction of MDA and H2O2 contents were found in the transgenic Arabidopsis plants comparing with WT under drought or salt stress.qRT-PCR analysis showed that overexpression of IbGATA24 up-regulated the genes involved in ABA and JA signalling pathway,proline synthesis pathway and ROS-scavenging system under salt and drought stresses.In addition,Y2H showed that IbSDT could interact with RLT1 proteinwhich regulated drought and salt stress tolerances together in plants.